Introduction

Over the last decade, numerous products containing high levels of caffeine have emerged [1,2]. These products include energy drinks, powdered caffeine, caffeine pills, buccal caffeine pouches, caffeinated peanut butter, and caffeine vaporizer sticks. These highly caffeinated products are disproportionally targeted to adolescents and young adults [3]. Of these products, the most widely used are highly caffeinated energy drinks, which come in a variety of different volumes (from 1.7 oz energy shots to 20 oz. cans) and caffeine concentrations (9–170 mg/oz.) [2,4,5]. Sales of energy drinks grew 60% from 2008 to 2013, illustrating the increased popularity and consumption of these beverages. Yet, increased accessibility of highly caffeinated products has coincided with increased reports of emergency departments visits because of energy drink consumption [6], highlighting the potential harms of exposure to highly caffeinated solutions to adolescents.

While the consumption of large quantities of caffeine itself is problematic [2,7], added health risks arise when caffeine is consumed with alcohol. It has been reported that 23% to 47% of adolescents and young adult alcohol users consume alcohol-mixed energy drinks [8,9]. Surveys of college-aged students suggest this population consumes large amounts of caffeine-mixed alcohol to fulfill hedonistic motives, such as increased pleasure from intoxication and increasing the intensity and/or nature of intoxication [10,11]. However, serious–and sometimes fatal–consequences can occur when mixing caffeine with alcohol [12–14]. While it is clear that consumption of caffeine-mixed alcohol solutions by adolescents and young adults carries a significant acute health risk, the long-term consequences of repeated exposures to caffeine-mixed alcohol are not yet well understood.

The lack of information on the potential long-term risks is particularly concerning given that adolescents, who are the predominant consumers of caffeine-mixed alcohol, are known to be more susceptible to changes in behavioral and neuronal adaptations from exposure to psychostimulants and drugs of abuse than adults [15–17]. Increased responses to cocaine-induced locomotor stimulation and reward have been observed in adolescent mice exposed to caffeine but not in animals exposed to caffeine in adulthood [17], suggesting chronic exposure outcomes in adolescence are not synonymous with exposures outcomes in adulthood. Legal and ethical issues surrounding alcohol use in minors heavily limits caffeine-mixed alcohol studies in human to self-reported survey-based results or in-laboratory performance tasks [18,19]; yet, animal studies provide a viable option for studying the effects of caffeine-mixed alcohol on adolescent behavior in a controlled setting [20]. Importantly, results observed in previous animal studies correlate with reported effects in adolescents and young adults [17,20–22]. Here we developed an animal model using adolescent mice to mimic exposure to caffeine-mixed alcohol as reported by college-aged adults [6,10,11].

Both caffeine and alcohol are known to increase dopamine release in dopaminergic reward pathways, specifically through their actions involving adenosine and dopamine receptors in the dorsal striatum and nucleus accumbens [23,24]. We hypothesized that repeated consumption of caffeine-mixed alcohol causes stronger activation of the dopaminergic reward pathway than caffeine or alcohol alone and could be on par with the levels of dopamine released by commonly abused psychostimulants, such as cocaine, leading to unique behavioral and pharmacological adaptations. To evaluate how chronic adolescent exposure to caffeine-mixed alcohol alters drug-related behaviors, we exposed C57BL/6 mice to caffeine-mixed alcohol throughout adolescence and monitored changes in locomotor sensitivity, ΔFosB accumulation, cocaine preference, cocaine sensitivity, and natural reward to saccharin. We observed unique behavioral and neurochemical effects of repeated caffeine-mixed alcohol exposure in adolescent mice that may indicate that these animals will experience future events involving caffeine-mixed alcohol, natural rewards, or cocaine and/or other psychostimulants differently than animals not exposed to caffeine-mixed alcohol in adolescence.

Materials and Methods

Animals

Adolescent (approximately postnatal day 28 [P28]) male and female C57BL/6 mice were obtained from Harlan Inc. (Indianapolis IN, USA) and allowed to acclimate for one week to handling and drug administration before behavioral testing began at postnatal day 35 [25,26]. Unless specified otherwise, mice were grouped housed in single grommet ventilated Plexiglas cages at ambient temperature (21°C) in a room maintained on a reversed 12L:12D cycle (lights off at 10.00, lights on at 22.00) in animal facilities, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Food and water were provided ad libitum and mice were not deprived of food or water at any time. All animal procedures were pre-approved by Institutional Animal Care and Use Committees of Purdue University and the University of California San Francisco and conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Locomotor sensitization via intraperitoneal exposure

Adolescent male and female C57BL/6 mice (n = 9–11 per group) were administered saline (0.9%), caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by intraperitoneal injection for either five days a week for two weeks (male only animals, Fig 1A) or four weeks (male and female animals, Fig 1B). Locomotor activity was measured for 60 minutes in locomotor activity boxes (L 27.3 cm x W 27.3 cm x H 20.3 cm, Med Associates, St Albans City VT, USA) immediately following drug administration on the days depicted in Fig 1A and 1B. Behavioral testing was conducted during the light cycle for each mouse. Mice were habituated to the behavioral testing room one-hour prior to acclimate to fan noise. To reduce the effect of novelty on locomotor activity, mice were habituated to the locomotor boxes the day before the first experiment.

Locomotor sensitization via oral gavage exposure

Adolescent male C57BL/6 mice (n = 6 per group) were administered water, caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by oral gavage for five days a week for four weeks (Fig 2). Locomotor activity was measured for 60 minutes in the locomotor activity boxes immediately following drug administration on the days depicted in Fig 2. Behavioral testing was conducted during the active/dark cycle for each mouse. Mice were habituated to the behavioral testing room one-hour prior to acclimate to fan noise. To reduce the effect of novelty on locomotor activity, mice were habituated to the locomotor boxes the day before the first experiment.

Male adolescent C57BL/6 mice were repeatedly exposed to exposed to water (H2O), 1.5 g/kg alcohol (ALC), 15 mg/kg caffeine (CAF) or caffeine-mixed alcohol (A+C), exposure by daily oral gavage (n = 6 per group) for 4 weeks for locomotor monitoring as depicted by the arrows. At the end of four weeks, animals were either perfused after one more drug administration (“IHC”) or subjected to behavioral tasks. Animals under “CPP” were subjected to cocaine conditioned place preference for cross-sensitization to cocaine reward. Animals in “SENS” were monitored for cocaine locomotor cross-sensitization. Natural reward consumption of saccharin was measured in “SACC” through four-hour limited-access, two-bottle choice between concentrations of saccharin (0.25, 0.5, 1.0, and 2.0 mM saccharin) and water for two days at each saccharin concentration.

Images were acquired via confocal microscopy (Nikon A1) at 20x magnification using an oil immersion objective. Gain and exposure were standardized to slices from a water-treated animal for proper control throughout image capture. For each animal, two images were collected, one image from the left hemisphere and one from the right hemisphere for the brain region of interest. Images were processed using ImageJ software (National Institutes of Health) for the number of ΔFosB positive cells in the dorsal striatum and shell of the nucleus accumbens per image. Positive cells were identified as areas with a specific intensity and area compared to background, as identified through Image J analysis. The total area of analysis for each images = 403072 um2.

Conditioned place preference to cocaine

Adolescent male C57BL/6 mice (n = 8–12 per group) were administered water, caffeine, alcohol, or caffeine-mixed alcohol via oral gavage, five days a week for four weeks as previously described (Fig 2). The following week, mice were conditioned to cocaine in a conditioned place preference paradigm (CPP, Fig 2 “CPP”) [28]. On day 1, mice were injected i.p. with saline and placed in a two-chamber conditioned place preference box (ENV-3013-2, Med Associates) to establish baseline preference the two chambers. Testing chambers contained unique tactile (wired mesh versus metal rod flooring) and visual (horizontal or vertical black and white striped wallpaper) cues for contextual usage to differentiate between the two chambers. Over the following eight conditioning days, mice received daily i.p. injection alternatively with saline or cocaine (1.5, 5, 15, or 30 mg/kg) and were confined for 30 minutes to either a cocaine-paired side or saline-paired side of the box in an unbiased approach. On the final day, saline was administered and the mice were placed in the CPP box in order to freely move between the two boxes for preference testing for 30 minutes (Fig 2). Preference was calculated as the difference in time spent in the cocaine-paired side between the pre- and post-conditioning tests. Mice that spent 70% of time in one side on the pre-conditioning day were excluded from the test. All conditioning was conducted during the dark/active cycle for each mouse.

Cocaine locomotor cross-sensitization

Adolescent male C57BL/6 mice (n = 7–8 per group) were administered water, caffeine (15 mg/kg), alcohol (1.5 g/kg), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol) by oral gavage for five days a week for four weeks (Fig 2). Locomotor activity was measured for 60 minutes in the locomotor activity boxes on the first and final day of drug administration. Locomotor activity was measured as described previously for 60 minutes following habituation to the testing room during the animals’ dark/active cycle. Three days after final drug administration, animals were injected with 0.9% saline (i.p.) and placed in the locomotor boxes for baseline locomotor activity for 60 minutes. Two days after this baseline measurement (total of 5 days since last drug treatment), animals were injected with 15 mg/kg cocaine (i.p.) and placed in the locomotor boxes for 60 minutes for total locomotor activity measurement (Fig 2 “SENS”).

Natural reward to saccharin

Natural reward was monitored through preference of sweet solution (saccharin) versus water in a four-hour, two bottle choice, drinking-in-the-dark paradigm [29] following adolescent exposure to drug solutions. Male adolescent C57BL/6 mice (n = 6–8 per group) were exposed to water or caffeine-mixed alcohol via oral gavage as described previously for four weeks in adolescence, shown in Fig 2. Upon final drug administration during the fourth week, animals were moved into single housing, double grommet cages for fluid consumption monitoring and to allow one weekend of acclimation to new cages. Three days after, saccharin solutions (0.25, 0.5, 1.0, 2.0 mM in reverse osmosis water) were prepared in 50 mL Falcon tubes, fitted with sippers, and distributed to the animals alongside a water control bottle during a four-hour, drinking-in-the-dark period to monitor saccharin consumption preference and volume (Fig 2 “SACC”) [30,31]. Bottles were added two hours into the dark cycle and removed four hours later, allowing behavioral testing during the animals’ active cycle. Weights of the bottles were measured to 0.1 gram. Each concentration was offered to the animals for two consecutive days before moving to the next concentration for total of eight days of drinking. The location of the water and saccharin bottles was reversed between days to prevent habit formation.

Statistical Analysis

All data are presented as means ± standard error of the mean. The analysis of pharmacological drug effects over time was performed using one-way or two-way, repeated measures ANOVA for adolescent drug treatment and time, followed by a Bonferroni post-hoc test to determine statistically significant differences between groups using GraphPad Prism5 software (GraphPad Software, La Jolla, CA, USA). Student’s unpaired t-test was used for analyzing less than two groups using GraphPad Prism5.

The locomotor sensitization we observed in adolescent mice exposed to caffeine-mixed alcohol resembled the locomotor sensitization commonly observed upon chronic cocaine exposure [32]. Chronic cocaine exposure is known to induce long-term increases in ΔFosB expression in the mesocortical and nigrostriatal dopaminergic pathways [33], thus we examined whether changes in ΔFosB expression occurred in the dorsal striatum and nucleus accumbens as a result of drug exposure (Fig 2). The shell of the nucleus accumbens was chosen (compared to nucleus accumbens core) as dopamine concentrations are known to preferentially increase in the shell following exposure to drugs of abuse [34]. One-way ANOVA analysis of these data was statistically significant for both dorsal striatum (F4, 29 = 17.43, p<0.0001, Fig 4A, 4C and 4D) and nucleus accumbens (F4, 28 = 10.73, p<0.0001, Fig 4B, 4C and 4E) indicating that treatment in general affected ΔFosB expression. Post-hoc analysis with Bonferroni’s multiple comparison test revealed that mice exposed to cocaine, caffeine, alcohol, or caffeine-mixed alcohol exhibited a significant increase in the number of ΔFosB positive cells in the dorsal striatum compared to water controls. Interestingly, mice exposed to caffeine-mixed alcohol or cocaine during adolescence, but not alcohol or caffeine alone, exhibited increased ΔFosB expression in the nucleus accumbens versus water controls.

Adolescent C57BL/6 mice (n = 6 per group) were repeatedly exposed by oral gavage to water (H2O), 15/mg/kg caffeine (CAF), 1.5 g/kg alcohol (ALC), caffeine-mixed alcohol (A+C), or 15 mg/kg cocaine (i.p., COC) for four weeks in adolescence, as shown in Fig 2. Three days after the final locomotor session, animals were exposed once more to their respective treatment. Brains were removed 30 minutes after exposure to last treatment via transcardial perfusion. Coronal brain slices were immunohistochemically stained for ΔFosB expression in the dorsal striatum (A, D) and nucleus accumbens (B, E), as indicated in C. All treatments increased ΔFosB accumulation in the dorsal striatum compared to water controls (A, D). Increases in ΔFosB accumulation were observed in the nucleus accumbens in animals exposed to caffeine-mixed alcohol compared to alcohol or caffeine alone (B, E). Quantification was achieved by counting the number of ΔFosB for each treatment using ImageJ software. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test, *, p<0.05; **, p<0.01, ***, p<0.0005, #, p<0.05, ###, p<0.0005; data represented as mean ± SEM.

We observed no difference in cocaine induced hyperlocomotion between water and caffeine-mixed alcohol exposed animals upon their first cocaine exposure during conditioning at any of the tested cocaine conditioning doses (S2A Fig). Additionally, there were no differences in 15 mg/kg cocaine induced locomotor activity during first conditioning session to cocaine between adolescent treatment groups (S2B Fig), suggesting that the attenuation in place preference observed in animals exposed to caffeine-mixed alcohol was not a result of alterations in locomotor response to cocaine. Adolescent exposure to caffeine-mixed alcohol also did not impact general locomotor activity during the pre-conditioning test day compared to water controls, although Bonferroni’s post-hoc analysis did show that caffeine exposed mice had significantly more locomotor activity than animals exposed to alcohol in adolescence (one-way ANOVA F3,29 = 4.976, p = 0.004, S2C Fig).